Staphylococcus aureus is an important human pathogen capable of causing infections in virtually all human tissues. The organism continues to cause significant morbidity and mortality in hospital and community settings despite huge investments in healthcare. The pathogenicity of this organism is dependent on its ability to produce various virulence factors, which are regulated by a large number of regulators forming a complex regulatory network. Elucidation of virulence regulation is crucial to understand pathogenesis of S. aureus. However, despite intensive efforts, how this pathogen coordinately controls its large number of virulence genes for a successful infection is still largely unknown. Thus, the long-term goal of this research is to understand virulence gene regulation in S. aureus. In our laboratory, we have employed the well-characterized capsule as a model virulence factor to understand virulence gene regulation in S. aureus. Capsule is an important virulence factor that endows the bacteria to resist host immune system but it also masks important cell surface components required for proper interactions of the bacteria with its host. Capsule therefore needs to be carefully regulated by the bacteria. We have previously characterized the cap operon responsible for capsule biosynthesis in detail at the molecular level. Subsequent studies conducted by us and others have shown that there are many regulators affecting capsule production. Interestingly, we found that several of these regulators are not typical transcriptional regulators suggesting that novel regulatory mechanisms are involved. In addition, many regulators affecting capsule have been shown to affect other virulence factors, such as toxins, suggesting that extensive interactions exists between regulatory networks of different virulence factors. Accordingly, in the first Specific Aim, we will study the fundamental mechanisms involved in capsule regulation by novel regulators using molecular and biochemical approaches. In the second Specific Aim, we will use genetic approaches to study how capsule is regulated by a network of regulators and how capsule and a-toxin, another important virulence factor, are differentially regulated by common regulators. Most virulence gene regulation studies have been conveniently carried out under laboratory conditions in vitro. Little is known about virulence regulation during an in vivo infection. Thus, n the last Specific Aim, we will carry out animal studies to understand virulence gene regulation in vivo using two very different animal models representing two different common diseases caused by S. aureus. We believe that successful completion of these proposed studies will provide new insights into virulence gene regulation in S. aureus thereby providing a firm basis to develop novel therapeutics to treat staphylococcal infections.